1. Field of the Invention
The present invention relates generally to the fields of immunopreventive therapy and vaccine development. More particularly, it concerns the development of novel adjuvants that can be used to generate more potent and robust vaccines. The application also describes the kinetic modulation of the immune response by these novel adjuvants.
2. Description of Related Art
Adjuvants for immunization are well known in the art and attempts to develop safe and effective adjuvants is an ongoing challenge. One of the main problems with current adjuvants is that most are unsuitable for use in human vaccines. One of the first adjuvant developed was Freund""s complete adjuvant which is a water-in-oil emulsion containing killed mycobacteria. This is unsuitable for use in humans due to severe side effects such as the lifelong persistence of oil in the tissues and the occasional production of sterile abscesses. Freund""s complete adjuvant has excellent immunopotentiating properties, but the side effects are so severe that it renders the use of this adjuvant unacceptable in humans (and sometimes in animals).
Other bacterially-derived adjuvants, such as MDP and lipid A are also associated with undesirable side-effects, and efforts are currently underway to develop benign analogs of these compounds (Roitt, 1994; Elgert, 1996). Oil based adjuvants in general are less desirable because they create undesirable side-effects such as visceral adhesions and melanized granuloma formations and because they cannot form a homogeneous mixture with DNA preparations such as DNA-based vaccines.
Aluminum salt-based adjuvants (such as alum) have excellent safety records but have the important disadvantages that they have a mediocre record in terms of efficacy with some antigens (Sjolander et al., 1998).
Oligonucleotides having unmethylated CpG dinucleotides have been shown to activate the immune system (A. Krieg, et al., 1995). CpG motifs may be inserted into a plasmid DNA vaccine vector, and replicated in bacteria thereby allowing the CpG motifs to retain their unmethylated form. As such, administration of a CpG adjuvant cloned into plasmid vectors would be simultaneous with the administration of a plasmid DNA vaccine. Alternatively, a CpG adjuvant in the form of free oligonucleotides may be administered before, during or after the administration of a plasmid DNA vaccine. Oligonucleotides having CpG motifs are modified at their phosphodiester linkages for stability purposes. For example, phosphodiester bonds in an oligonucleotide may be replaced by phosphorothioate linkages. DNA oligonucleotides containing unmethylated CgG dinucleotides are currently in phase I human trials. One note of caution is that the phosphorothioate oligonucleotide backbone, while essential for nuclease resistance, also confers a long half life and the long-term effects of maintaining these modified compounds in the body are yet to be determined.
The delivery system referred to as ISCOM contains the adjuvant saponin. Crude saponins are toxic, and much effort has gone towards purifying a less toxic saponin fraction which still retains the adjuvant properties. Results with a semi-purified fraction termed Quil A have been inconsistent, with differing levels of toxicity observed with different preparations. ISCOMs have now progressed to phase I and II of human trials, so the long term effects of these compounds remain to be seen (Sjolander et al., 1998). An exhaustive list of adjuvants are also described in Stewart-Tull, 1994.
Other immunomodulatory compounds have been proposed, including 8-bromoguanosine and ganglioside GM2 (Goodman, 1983; Livingston, 1995; Scheuer et al., 1985). Synthetic guanine nucleosides derivatized at either the C8 or at both N7 and C8 positions of the guanine base have been shown to possess some immunostimulatory and immunomodulatory properties (Goodman and Goodman, 1994; Reitz et al., 1994). None of these have yet been determined to possess the necessary characteristics of stability, safety and efficacy for clinical use as immune adjuvants in humans.
In summary, it appears that current adjuvant technology is still limited, as reflected by the fact that alum is the only FDA-approved human vaccine adjuvant.
The present invention overcomes the limitations of the prior art. The inventor has shown that two bacterial nucleotides, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), can modulate the host immune system to mount a rapid immune response against a specific antigen. Co-administration of an antigen, alum, ppGpp, pppGpp or a mixture of ppGpp and pppGpp dramatically decreases the time required to elicit an immune response against the antigen. These may be co-administered as follows; 1) antigen and ppGpp; or antigen and pppGpp; or antigen and a mixture of ppGpp and pppGpp; 2) or antigen and ppGpp and alum; or antigen and pppGpp and alum; or antigen and a mixture of ppGpp and pppGpp and alum. It is expected that these and related agents may have broad use as adjuvants, as stimulants of the innate immune response, and as modifiers of the character of an immune response.
The bacterial nucleotides guanosine tetra- and penta-phosphate have never been tested as immunologic adjuvants. The present invention demonstrates that these compounds have an immunomodulatory effect in a mouse host when co-administered with an antigen in the presence of alum. An unexpected and desirable feature of the immune response is the increased kinetic rate of antibody elicitation as compared with standard alum adjuvant. Given that guanosine tetra- and pentaphosphate are small mononucleotides, it is likely that they exert their immunomodulatory effects by macrophage activation rather than by acting as repository adjuvants. The mechanism by which (p)ppGpp exerts an immunomodulatory effect is not yet fully understood.
The dramatic increase in the kinetic response that was observed in this study indicates that (p)ppGpp may be affecting the immune system in a way that differs from other adjuvants. To date, the main use of adjuvants has been to improve the magnitude of antibody responses (immunostimulatory effect). The identification of (p)ppGpp as a kinetic modulator of immune response (immunomodulatory effect) could have profound effects in the field of vaccinology. This novel and unexpected effect of (p)ppGpp would allow its use as a co-adjuvant, in combination with adjuvants that act by a different mechanism. It is contemplated that the nucleotides of this invention may be used in compositions that can modulate immune responses, in addition to any other adjuvant. An exhaustive list of existing adjuvants is described in Stewart-Tull, 1994, and can be found by one of ordinary skill in the art.
The invention provides a composition comprising a nucleotide that is capable of modulating the host immune system. The nucleotides of the invention modulate the immune system by altering the kinetics of response of the host immune system. In preferred embodiments, the nucleotides are guanosine tetraphosphate (ppGpp), guanosine pentaphosphate (pppGpp), or a mixture of ppGpp and pppGpp. In particular embodiments, administration of compositions comprising one or more of these nucleotides provides a more rapid response of the host immune system to antigens.
In another aspect of the invention, the composition further comprises alum. In certain embodiments of the invention, the composition further comprises an antigen.
The skilled artisan will realize that ppGpp and pppGpp may be chemically modified to enhance their stability and/or efficacy. Such modified nucleotides may still be used within the scope of the present invention. In non-limiting embodiments, labile phosphodiester or phosphoester linkages may be replaced with more stable linkages, such as phosphorothioate or thioester linkages. In certain embodiments, the nucleotides comprise a guanine residue. In preferred embodiments, the guanine residue is not chemically modified or otherwise substituted.
A further aspect of the invention comprises the administration of a nucleotide adjuvant. In one embodiment of the invention, the nucleotide adjuvant comprises guanosine tetraphosphate (ppGpp). In another embodiment of the invention, the nucleotide adjuvant comprises guanosine pentaphosphate (pppGpp). In still another embodiment of the invention, the adjuvant comprises a mixture of guanosine tetraphosphate and guanosine pentaphosphate.
Another further aspect of the invention comprises the administration of a nucleotide adjuvant in combination with an antigen. In one embodiment of the invention, the adjuvant is administered before administration of the antigen. In yet another embodiment of the invention, the adjuvant is administered after administration of the antigen. In still another embodiment of the invention, the adjuvant is administered in conjunction or substantially simultaneously with the antigen.
The invention further provides a method of producing antibodies by administering a nucleotide adjuvant. In one aspect of the invention, the nucleotide adjuvant comprises ppGpp. In another aspect of the invention, the nucleotide adjuvant comprises pppGpp. In a further embodiment of the invention the nucleotide adjuvant, comprises a mixture of guanosine tetraphosphate and guanosine pentaphosphate.
In one aspect of the invention, the method of producing antibodies further comprises administration of alum. In a further embodiment of the invention, the method of producing antibodies further comprises the administration of an antigen. In one other embodiment of the invention, the method of producing antibodies comprises administration of the adjuvant before administration of the antigen. In still another embodiment of the invention, the method of producing antibodies comprises administration of the adjuvant after administration of the antigen. In yet another embodiment of the invention, the method of producing antibodies comprises administration of the adjuvant in conjunction or substantially simultaneously with the antigen.
The invention also provides an antibody produced by the method of administering a nucleotide adjuvant. In one aspect of the invention, the nucleotide adjuvant comprises guanosine tetraphosphate (ppGpp). In another aspect of the invention, the nucleotide adjuvant comprises guanosine pentaphosphate (pppGpp). In a further embodiment of the invention, the nucleotide adjuvant comprises a mixture of guanosine tetraphosphate and guanosine pentaphosphate.
In another embodiment of the invention, the antibody is produced by further administering alum. In certain embodiments of the invention, the antibody is produced by further administration of an antigen. In a related embodiment of the invention, the antibody is produced by further administration of the adjuvant before administration of the antigen. In another related embodiment of the invention, the antibody is produced by further administration of the adjuvant after administration of the antigen. In yet another related embodiment of the invention, the antibody is produced by further administration of the adjuvant in conjunction or substantially simultaneously with the antigen.
In preferred embodiments, the pharmaceutical formulations suitable for administration of the nucleotides and adjuvants developed in this invention are described. Pharmaceutical formulations for different vaccine types that may be used in the present invention are described. Preferred modes of delivery of the nucleotides, adjuvants and vaccines are also described.
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Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.